Polypeptides having cellulase activity

Abstract

The present disclosure relates to CBH II chimera fusion polypeptides, nucleic acids encoding the polypeptides, and host cells for producing the polypeptides.

What is claimed is: 
     
       1. A recombinant variant of a parent cellulase comprising the sequence of SEQ ID NO:2, 4, 12, or 64-66 wherein the residue corresponding to position 311 of SEQ ID NO:4 is substituted with serine in said variant, wherein the variant has cellulase activity and increased thermostability compared to the parent cellulase lacking a serine at the same position. 
     
     
       2. A recombinant polypeptide comprising a sequence that (i) is between 95% to 99% identical to SEQ ID NO:13 comprising a C312S; (ii) is between 95% to 100% identical to SEQ ID NO:14 comprising a C314S; (iii) is between 95% to 100% identical to SEQ ID NO:15 comprising a C315S; (vi) is between 95% to 100% identical to SEQ ID NO:16 comprising a C313S; (v) is between 95% to 100% identical to SEQ ID NO:17 comprising a C311S; (vi) is between 95% to 100% identical to SEQ ID NO:19 comprising a C313S; (vii) is between 95% to 100% identical to SEQ ID NO:21 comprising a C312S; or (viii) is between 95% to 100% identical to SEQ ID NO:22 comprising a C31 S wherein the foregoing polypeptides have cellulase activity and improved thermostability compared to their corresponding parental (wild-type) protein lacking a Cys→Ser mutation. 
     
     
       3. A recombinant polypeptide, comprising a sequence selected from the group consisting of:
 (a) a polypeptide having greater than 97% identity to a sequence selected from the group consisting of: (i) SEQ ID NO:13 and having a Ser at position 312, (ii) SEQ ID NO:14 and having a Ser at position 314, (iii) SEQ ID NO:15 and having a Ser at position 315, (iv) SEQ ID NO:16 and having a Ser at position 313 (v) SEQ ID NO:17 and having a Ser at position 311, (vi) SEQ ID NO:19 and having a Ser at position 313, (vii) SEQ ID NO:21 and having a Ser at position 312, and (viii) SEQ ID NO:22 and having a Ser at position 311, and wherein each of the foregoing polypeptides have cellulase activity; and 
 (b) a chimeric polypeptide comprising at least two domains from two different parental cellobiohydrolase polypeptides, wherein the domains comprise from N- to C-terminus: (segment 1)-(segment 2)-(segment 3)-(segment 4)-(segment 5)-(segment 6)-(segment 7)-(segment 8); wherein: 
 segment 1 comprises a sequence that is at least 95-100% identical to amino acid residue from about 1 to about x 1  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); segment 2 comprises a sequence that is at least 95-100% identical to amino acid residue x 1  to about x 2  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); segment 3 comprises a sequence that is at least 95-100% identical to amino acid residue x 2  to about x 3  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); segment 4 comprises a sequence that is at least 95-100% identical to amino acid residue x 3  to about x 4  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); segment 5 comprises a sequence that is at least 95-100% identical to about amino acid residue x 4  to about x 5  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); segment 6 comprises a sequence that is at least 95-100% identical to amino acid residue x 5  to about x 6  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); segment 7 comprises a sequence that is at least 95-100% identical to amino acid residue x 6  to about x 7  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); and segment 8 comprises a sequence that is at least 95-100% identical to amino acid residue x 7  to about x 8  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); 
 wherein x 1  is residue 43, 44, 45, 46, or 47 of SEQ ID NO:2, or residue 42, 43, 44, 45, or 46 of SEQ ID NO:4 or SEQ ID NO:6; x 2  is residue 70, 71, 72, 73, or 74 of SEQ ID NO:2, or residue 68, 69, 70, 71, 72, 73, or 74 of SEQ ID NO:4 or SEQ ID NO:6; x 3  is residue 113, 114, 115, 116, 117 or 118 of SEQ ID NO:2, or residue 110, 111, 112, 113, 114, 115, or 116 of SEQ ID NO:4 or SEQ ID NO:6; x 4  is residue 153, 154, 155, 156, or 157 of SEQ ID NO:2, or residue 149, 150, 151, 152, 153, 154, 155 or 156 of SEQ ID NO:4 or SEQ ID NO:6; x 5  is residue 220, 221, 222, 223, or 224 of SEQ ID NO:2, or residue 216, 217, 218, 219, 220, 221, 222 or 223 of SEQ ID NO:4 or SEQ ID NO:6; x 6  is residue 256, 257, 258, 259, 260 or 261 of SEQ ID NO:2, or residue 253, 254, 255, 256, 257, 258, 259 or 260 of SEQ ID NO:4 or SEQ ID NO:6; X 7  is residue 312, 313, 314, 315 or 316 of SEQ ID NO:2, or residue 309, 310, 311, 312, 313, 314, 315 or 318 of SEQ ID NO:4 or SEQ ID NO:6; and x 8  is an amino acid residue corresponding to the C-terminus of the polypeptide have the sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, 
 wherein the chimeric polypeptide comprises a Ser at position corresponding to position 314 of SEQ ID NO:2 or position 311 of SEQ ID NO:4 and wherein the chimeric polypeptide has cellulase activity and improved thermostability and/or pH stability compared to a CBH II polypeptide comprising SEQ ID NO:2, 4, or 6. 
 
     
     
       4. The recombinant polypeptide of  claim 3 , wherein segment 1 comprises amino acid residue from about 1 to about x 1  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); segment 2 is from about amino acid residue x 1  to about x 2  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); segment 3 is from about amino acid residue x 2  to about x 3  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); segment 4 is from about amino acid residue x 3  to about x 4  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); segment 5 is from about amino acid residue x 4  to about x 5  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); segment 6 is from about amino acid residue x 5  to about x 6  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); segment 7 is from about amino acid residue x 6  to about x 7  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”); and segment 8 is from about amino acid residue x 7  to about x 8  of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”). 
     
     
       5. An enzymatic preparation comprising a polypeptide of  claim 1 ,  2  or  3 . 
     
     
       6. A method of treating a biomass comprising cellulose, the method comprising contacting the biomass with a polypeptide of  claim 1 ,  2  or  3 . 
     
     
       7. A method of treating a biomass comprising cellulose, the method comprising contacting the biomass with an enzymatic preparation of  claim 5 .